PROJECT SUMMARY/ABSTRACT Gut microbiome is a dynamic and malleable microbial community vital to the maintenance of human and animal health. However, under the influence of a calorically rich Western-type diet, a healthy gut microbiome can transform into a dysfunctional state promoting the onset and progression of several non-communicable chronic diseases, including atherosclerosis. A central unresolved question had been whether and how it might be possible to remodel a dysfunctional gut microbiome within a living human or animal to treat or prevent disease progression. We now show that this indeed can be achieved. Building on our recent discoveries and advances highlighted here, the proposed multidisciplinary research program is aimed at developing novel chemical agents, advanced screening methods, and predictive bioinformatic tools to help identify and validate the biological mechanisms driven by an imbalanced gut microbiome that fuel chronic inflammation and progression of atherosclerosis. We provide Preliminary Results data to show, for the first time, that a dysfunctional gut microbiome induced by a Western diet (WD) can be selectively remodeled in vitro and in vivo to prevent the development of atherosclerosis. We disclose that self-assembling eight-residue cyclic D,L-a-peptides selected from a novel en masse in vitro screening protocol can function as bacterial growth modulators and in a targeted manner remodel a WD-induced dysfunctional gut microbiome in vivo to prevent development of atherosclerosis in LDLr-/- mice. Directed remodeling of the gut microbiome following 10-week daily oral administration of two lead peptides to mice caused diverse and beneficial biological effects in the host, including marked reductions in plasma total cholesterol levels and atherosclerotic plaques, extensive reprogramming of the microbiota and host transcriptomes, increased populations of intestinal Helios+ Treg immune cells, suppressed the production of a number of pro-inflammatory cytokines (including IL-6, TNF-a, and IL-1b), improved gut barrier integrity, and rebalanced levels of disease-relevant metabolites, such as short-chain fatty acids (SCFAs) and bile acids. Building on these advances, the proposed studies seek to improve and exploit our in vitro method for compound screening against gut microbial populations as a whole to identify molecules that can selectively modulate the overall microbiome composition without significantly reducing species diversity/richness. Complementary to the screening efforts are a host of mechanistic studies carried out in animal models of atherosclerosis. Comparative analyses of the microbial metagenomics and host transcriptomics for treated vs. control animals would provide data for developing improved compound scoring and categorization tools. Our research program focuses on characterizing and manipulating the microbiome in its entirety, and this approach could provide new tools and methods for exploring host/microbiome interactions, interrogating specific mechanistic questions, and identifying novel pathways and therapeutic targets.